The studies described in this proposal are designed to investigate the molecular details of the interactions within the Negative Regulatory Region (NRR) of the Notch receptor that acts as the activation switch for this evolutionarily conserved biosignaling pathway. They aim to provide the structural insight needed to validate or refute the multitude of current models in literature, as well as test novel models that attempt to explain how the Notch receptor is ensured in an "OFF" state prior to ligand binding under normal conditions, as well as how Notch activation becomes ligand independent in cancer associated mutations. The two structural domains that make up the single functional "activation switch" for the Notch receptor share an extensive inter-domain interface, but also possess the capacity to exist in isolation. The three tandem LIN-12/Notch-Repeats (LNRs) keep the receptor in its resting conformation prior to ligand binding by forming a steric plug to conceal the key metalloprotease cleavage site within the heterodimerization (HD) domain. Since dysregulated Notch signaling has been implicated in a diverse spectrum of human diseases, a molecular understanding of these two protein domains at the amino acid level will greatly benefit ongoing pharmaceutical initiatives. Since the LNRs represent a novel category of small disulfide rich protein modules that are also found in other multi-domain proteins with completely different biological functions, the studies proposed under the first specific aim will also have a broader impact on the biomedical field by enhancing our understanding of how individual amino acids impact the structure and stability of protein domains and providing additional insight into the decades long protein folding problem. The second aim of this proposal, on the other hand, will focus on evaluating how individual cancer mutations impact the structural integrity and stability of the HD domain, an area that has been gaining a rapid appreciation since the discovery of the high frequency of cancer-associated mutations that map to the HD Domain of human Notch1 five years ago. Already inhibition of Notch signaling is under investigation as a novel alternative for the treatment of several malignancies in which activation of Notch signals has an oncogenic role and hence the findings of this project can potentially have immediate therapeutic impact. In addition, since the proposed studies will use a wide spectrum of biochemical and biophysical tools to address the two specific aims and are multidisciplinary and collaborative in nature, they will provide ample opportunities for many enthusiastic and motivated undergraduates to apply fundamental physicochemical principles to current biomedical problems. PUBLIC HEALTH RELEVANCE: Several lines of evidence indicate that aberrant regulation of Notch activity plays a major role in human cancers. The Negative Regulatory Region of the Notch protein contains two domains that together acts as the "activation switch" for this critical biosignaling pathway. This research will help to provide a molecular insight into the structural specifics of the two protein units within this region and ultimately help the design of therapeutic interventions to treat cancer patients.